Controlling Gene Transfer with GeneGuard
Background Many people view gene transfer between organisms as being positive, leading to adaptations in organisms that would otherwise not occur. However, there are many examples where horizontal gene transfer leads to undesirable outcomes, specifically in the case of synthetically constructed DNA. The release of genetically modified microbes and their DNA into the environment can lead to something called "genetic pollution." Many microbes are only meant to exist within a bioreactor and are therefore not a concern. However, there are other types of modified bacteria that are not contained within a reactor, for example, bacteria involved in bioremediation or biosensing. Situations like this, where bacteria are used directly in the environment can lead to unwanted genetic pollution if uncontrolled. Genetic pollution is a concern because it may lead to the spread of unwanted genes (via horizontal gene transfer) to wild type organisms. Alterations such as antibiotic resistance can have a profound effect on bacterial populations, so it is important to ensure these types of genes are controlled and do not make it into the environment. Researchers have developed plasmid systems designed with biosafety in mind. The plasmid system is called GeneGuard was developed in an attempt to be nearly impossible for any type of bacteria except the original host to use. The product has three control systems that, although they do not prevent horizontal gene transfer, help prevent the plasmid from ever surviving within a different host.Wright, Oliver, et al. "[http://pubs.acs.org/doi/abs/10.1021/sb500234s GeneGuard: a Modular Plasmid System Designed for Biosafety]." ''ACS synthetic biology'' (2014). GeneGuard 3-part Security System Conditional Origin of Replication The first method of preventing the GeneGuard plasmid from being expressed in an organism other than the host is its conditional origin of replication. This approach involves putting the gene that controls for the production of the protein responsible for initiating plasmid replication on the chromosome. This approach prevents the plasmid from replicating if the host doesn't contain that specific replication gene, making the plasmid extremely specific to the single host that the researcher chooses. Another advantage to controlling the origin of replication is that researchers can tune the copy number of the plasmid by changing the gene responsible for replication. This effectively allows for increased or decreased expression of the plasmid as desired. Rich-Media Compatible Auxotrophies The next security feature of the GeneGuard system is a host induced auxotrophy that the plasmid is capable of fixing. This means that within the genome of the host, a specific gene required for survival has been knocked out. The plasmid holds the gene that fixes the auxotrophy, meaning that it produces the protein that has been knocked out in the genome. In this case, the auxotrophy security measure prevents the host from being able to survive without the plasmid. Toxin-Antitoxin Pairs Finally, in the GeneGuard system, there exists a toxin-antitoxin pair that only allows growth when both are present. More specifically, there is a toxin gene on the plasmid that will prevent it from surviving in any host without the antitoxin on the chromosome. This is another measure to ensure that this plasmid is not capable of surviving outside of the original host. The selected toxin is a very broad range toxin, capable of killing many types of bacteria that don't contain the corresponding antitoxin gene. Importance Horizontal gene transfer is a very important mechanism in evolution that allows for the transfer of genetic material that will sometimes allow the bacteria to be better adapted for survival. Some common adaptations include antibiotic resistance (as discussed above), or the transfer of virulence factors that allow bacteria to compete better within a specific environment. Such adaptations in virulence have been shown to give the parasite an increased ability to survive. de Roode, Jacobus C., et al. "[http://www.pnas.org/content/102/21/7624.short Virulence and competitive ability in genetically diverse malaria infections]." ''Proceedings of the National Academy of Sciences of the United States of America'' 102.21 (2005): 7624-7628. Clearly, it is important to tightly control the gene transfer between organisms and to prevent unwanted genetic pollution. References